Adoptive T cell immunotherapy strives to harness the power and specificity of the immune system to cure cancer in human patients. This approach relies on the transfer of tumor-reactive CD8+ T cells into patients, but the anticipated clinical benefits have not been achieved due to the induction of T cell tolerance. Peripheral tolerance is a multifaceted process, characterized by compromised proliferation, impaired effector function, and deletion of autoreactive T cells. Efforts to overcome tolerance for improved immunotherapy have been hampered because the T cell intrinsic pathways that regulate whether tumor/self-reactive T cells are directed toward tolerance versus immunity have yet to be defined. Using a pre-clinical mouse model of T cell tolerance, we found that Listeria monocytogenes vaccination at the time of adoptive CD8+ T cell transfer prevents tolerance. Specifically, transferred tumor/self-reactive T cells acquired effector function, displayed increased in vivo persistence, and provided a survival benefit to tumor-bearing hosts receiving adoptive T cell immunotherapy. T cell rescue corresponded with expression of the transcription factors T-bet and Eomesodermin (Eomes), both of which failed to be induced under tolerizing conditions in the absence of vaccination. T-bet and Eomes have a defined role in dictating type-I (IFN-? mediated) immune responses. Aside from their role in promoting IFN-? expression, these transcription factors also influence expression of several other genes that encode effector molecules important for CD8+ T cell trafficking or control of infected or malignant cells, includin CXCR3, granzyme B, perforin, and FasL. Our data suggest that T-bet and Eomes may be fundamental to the decision of T cell immunity versus tolerance, and as such, we propose to decipher the role of these transcription factors in this important fate decision. To this end, we wll utilize mice with genetic modulation of T-bet and Eomes in peripheral tumor/self-reactive CD8+ T cells as tools to dissect the role of these transcription factors during induction of T cell tolerance and under conditions of vaccine-mediated T cell rescue. Specific Aim I will focus on the individual and collective contributions of T-bet and Eomes to vaccine-mediated rescue of peripheral CD8+ T cell tolerance. We will evaluate effector cytokine production, T cell persistence, and transcript and protein production for T-bet or Eomes target genes in T cells rendered unable to upregulate T-bet, Eomes, or both; or T cells with constitutive T-bet expression. Specific Aim II will then focus on characterizing the specific contributions of these molecules to providing anti-tumor immunity within a tolerizing environment. Survival of tumor-bearing hosts will be used as a readout for therapeutic efficacy of adoptive immunotherapy with CD8+ T cells rendered genetically unable to express T-bet, Eomes or both; or T cells with constitutive T-bet expression. Identifying the contributions of T-bet and Eomes to T cell dysfunction during CD8+ T cell tolerance and adoptive immunotherapy may lead to improvements in T cell-based therapies for patients with cancer.